Aliyu Proposal

 

Molecular characterization of Quinolone resistance in Salmonella enterica serovar typhi using sequencing of the gyrA quinolone resistance determining region (QRDR)

 

 

PRINCIPAL INVESTIGATOR - DR. ALIYU, SHAMSUDIN

Department of Medical Microbiology,

Ahmadu Bello University,

Zaria

DR. ALIYU, SHAMSUDIN

Molecular characterization of Quinolone resistance in Salmonella enterica serovar typhi using sequencing of the gyrA quinolone resistance determining region (QRDR)

ABSTRACT

Introduction: Typhoid fever is a major public health problem in developing countries.  Increasing drug resistance of Salmonella enterica serovar typhi to many antibiotics is a global concern. Resistance to fluoroquinolones is of serious concern because ciprofloxacin is the drug of choice for treating invasive human salmonellosis.  Alteration caused by a single mutation within the quinolone resistance –determining region (QRDR) of DNA qyrase subunit of gyrA is the main mechanism behind quinolone resistance.

Aims: The study aims to define the antimicrobial resistance profile of isolates of Salmonella typhi to ciprofloxacin, ampicillin, co-trimoxazole, chloramphenicol, and ceftriaxone; and to also correlate nalidixic and ciprofloxacin minimum inhibitory concentrations (MICs) to mutations in the gyrA gene.

Methods:  Salmonella typhi strains consecutively isolated over an 8 month period from the blood and stool culture benches of the medical microbiology laboratory of Ahmadu Bello University Teaching Hospital, Zaria, over; will be studied. Antimicrobial susceptibility will be carried out using the disk diffusion method. The minimum inhibitory concentrations (MICs) of a quinolone (Nalidixic acid) and a fluoroquinolone (Ciprofloxacin) will be used to characterise resistance of salmonella typhi isolates to quinolones. Polymerase chain reaction (PCR) and nucleotide sequencing of the gyrA gene will be utilized to characterise mutations in this gene.

 

S/N

ITEM/SERVICE

QUANTITY

UNIT COST (N)

TOTAL (N)

 

Antimicrobial Disks (50 disks cartridge)

10

4,400

44,000

1

Etest Strips (30 per Pack)

4

45,000

180,000

2

Mueller Hinton Agar

1

15,000

15,000

3

Mac Conkey Agar

1

15,000

15,000

4

Microbact 12A Identification Kit (60 Tests)

1

49,000

49,000

5

Microbact Reagent set

1

20,300

20,300

6

Mineral Oil for Microbact

1

4,200

4,200

7

Oxidase Strip for Microbact

1

6,600

6,600

8

Escherichia coli ATCC® 25922 Control Strain

1

46,408.00

46,408.00

9

Salmonella enterica subsp. typhi ATCC®  6539 control Strain

1

46,939.71

46,939.71

10

Petri disk(500 per Carton)

1

15,000

15,000

11

Plain Sterile tube (100 per pack)

1

2,500

2,500

12

PCR Amplification of  gyrA gene

60

3,500

210,000

13

Sequencing of  gyrA gene

60

4,500

270,000

14

PCR Primers

2

2,300

4,600

 

 

 

 

 

 

TOTAL  COST

 

 

929,547.71

BUDGET

 

BUDGET JUSTIFICATION

An estimated 50 to 60 isolated will be collected and processed over an 8 month period. This is based on review of records of the blood and stool culture benches of the department of medical microbiology of Ahmadu Bello University Teaching Hospital, Zaria.

Antibiotic Disks (50 disks per cartridge) - Two each of ciprofloxacin, ampicillin, co-trimoxazole, chloramphenicol, and ceftriaxone will be required.

Etest Strips (30 per Pack) - Two packs each of Nalidixic and ciprofloxacin required to determine MICs to quinolones.

Mueller Hinton Agar - Required for Antimicrobial susceptibility testing

Mac Conkey Agar - Required for cultivation of salmonella typhi isolates.

Microbact 12A Identification Kit (60 Tests) - Used for biochemical identification of salmonella typhi isolates

Microbact Reagent set - Reagents required for use with Microbact identification kit.

Mineral Oil - Reagent required for use with Microbact identification kit.

Oxidase Strips - Reagent required for use with Microbact identification kit.

Escherichia coli ATCC® 25922 Control Strain - Used as control in antimicrobial susceptibility testing

Salmonella enterica subsp. typhi ATCC® 6539 control Strain - Used as positive control for identification kits and as a positive control for PCR.

Petri dish (500 per Carton) – Will be used for preparation of Mueller Hinton Agar and Mac Conkey Agar

Plain Sterile tube (100 per pack) - To be used to prepare inoculum for antimicrobial susceptibility test

PCR Amplification of gyrA gene - This will be carried out at DNA Labs, in Kaduna State

Sequencing of gyrA gene - Sequencing of the gyrA will be carried out at Inqaba biotech in Ibadan.

PCR Primers - Used for amplification of gyrA during Polymerase Chain Reaction

 

Project Narrative:

This study will build capacity in the use of molecular techniques such as nucleotide sequencing in research. The study will also set the background for the use of bacterial whole genome sequencing to characterise antibiotic resistance in bacteria.

 

RESEARCH PLAN

I. Specific Objectives

1. To evaluate the antimicrobial susceptibility pattern of salmonella typhi isolates to ciprofloxacin, ampicillin, co-trimoxazole, chloramphenicol and ceftriaxone using the disk diffusion method.

2. To classify quinolone resistance in salmonella typhi isolates using the Minimum inhibitory concentrations (MICs) of a quinolone (Nalidixic acid) and a fluoroquinolone (Ciprofloxacin).

3. To detect and characterise the molecular bases of quinolone resistance in salmonella typhi isolates through amplification and sequencing of the gyrA gene.

II. Background and Significance

Typhoid fever is a major public health problem in developing countries.1,2 Increasing drug resistance of Salmonella enterica serovar typhi to many antibiotics is a global concern.3 Resistance to fluoroquinolones is of serious concern because ciprofloxacin is the drug of choice for treating invasive human salmonellosis.4  Alteration caused by a single mutation within the quinolone resistance –determining region (QRDR) of DNA qyrase subunit of gyrA is the main mechanism behind quinolone resistance.5

III. Preliminary Data - No preliminary data

IV. Experimental Design and Methods

Twenty five Salmonella typhi strains consecutively isolated from blood and stool culture benches of the medical microbiology laboratory of Ahmadu Bello University Teaching Hospital, Zaria; will be studied. Microbact Kit (oxoid) will be used for identification of salmonella typhi isolates. Antimicrobial susceptibility will be carried out using the Etest method, according to CLSI guidelines.6 The minimum inhibitory concentrations (MICs) of a quinolone (Nalidixic acid) and a fluoroquinolone (Ciprofloxacin) will be used to characterise resistance to quinolones in salmonella typhi isolate.7 Amplification of the gyrA gene will be carried out using Polymerase chain reaction (PCR) according to methods described by Dimitrov et al.8 Gene sequencing will be carried at Inqaba biotech (Ibadan) using Sanger sequencing. Data from nucleotide sequencing of the gyrA gene will be utilized to characterise mutations in this gene, according to methods described by Dimitrov et al.8 and Kariuki et al9.

 

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References

1. Mogasale V, Maskery B, Ochiai RL, et al. Revisiting the burden of typhoid fever in low-

               

    and middle-income countries for policy considerations. Lancet Glob Health 2014; 2: e570– 

 

    80.

 

2. Crump JA, Lubsy SP, Mintz ED. The global burden of enteric fever. Bull World Health

 

    Organ 2004; 82: 346–53.

 

3.  Bhan, M. K., R. Bhal, and S. Bhatnagar. 2005. Typhoid and paratyphoid

 

    fever. Lancet 366:749–762

4. Kariuki S, Gordon MA, Feasey N, Parry CM. Antimicrobial resistance and management

    of invasive Salmonella disease. Vaccine. 2015 Jun 19;33 Suppl 3:C21-9.

5. Hirose K, Hashimoto A, Tamura K et al. DNA sequence analysis of DNA gyrase and

 

    DNA topisomerase IV quinolone resistance-determining regions of Salmonella enterica

 

    serovar Typhi and serovar Paratyphi A. Antimicrob Agents Chemother 2002; 46: 3249–52.

 

6. Clinical Laboratory and Standards Instituite (CLSI). Performance Standards for

    antimicrobial susceptibility testing; twenty first informational supplement. Wayne,

    Pensylvania: author, 2012. CLSI document M100-S22.   

7. Bae DH, Baek HJ, Jeong SJ, Lee YJ. Amino acid substitutions in gyrA and parC    

    associated with quinolone resistance in nalidixic acid-resistant Salmonella isolates. Ir Vet   

    J. 2013:15;66(1):23.

8. Dimitrov T, Dashti AA, Albaksami O, Udo EE, Jadaon MM, Albert MJ. Ciprofloxacin-

    resistant Salmonella enterica serovar typhi from Kuwait with novel mutations in gyrA and

    parC genes. J Clin Microbiol. 2009;47(1):208-11

9. Kariuki S, Revathi G, Muyodi J, Mwituria J, Munyalo A, Mirza S, Hart CA.

    Characterization of multidrug-resistant typhoid outbreaks in Kenya. J Clin Microbiol. 2004;

    42(4):1477-82.

 

Plan for protection of Human Subjects – Not Applicable

 

Project Mentor -

Professor A. T. OLAYINKA (M.B, B.S; FMCPath, MPH)

Consultant Clinical Microbiologist,

Ahmadu Bello University,

Zaria, Nigeria.

 

Pending IRB Approval – Not Applicable